A bovine platelet lysis buffer, its preparation method and application

Abstract

This invention belongs to the field of cell biology, specifically relating to a bovine platelet lysate (bPL), its preparation method, and its applications. This invention separates bovine peripheral blood platelets using a double centrifugation method, and employs repeated freeze-thaw cycles, ultrasonic disruption, and Ga... 2+ The activation method was used for lysis, and the effects of different lysis methods on growth factor release and total protein content were compared. Results showed that all three methods could successfully prepare bPL. The examples demonstrated that the bPL-containing composite culture system described in this invention exhibited good performance in terms of cell proliferation rate, colony formation ability, and long-term culture stability, with overall effects approaching those of traditional serum culture systems. The bPL culture system showed good proliferative capacity and adipogenic and osteogenic differentiation potential in mesenchymal stem cell culture. It demonstrated superior or near-superior performance in promoting cell proliferation and reducing cell senescence compared to the fetal bovine serum culture system, indicating that bPL has good application potential in the in vitro culture of mesenchymal stem cells.

Description

Technical Field

[0001] This invention belongs to the field of cell biology, specifically relating to a bovine platelet lysis buffer, its preparation method, and its application. Background Technology

[0002] Platelet lysate (PL) is a blood-derived product obtained by lysing platelets in vitro to release their intracellular bioactive substances. Platelet α-granules are rich in various regulatory factors, including platelet-derived growth factor (PDGF), transforming growth factor β (TGF-β), vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and insulin-like growth factor (IGF-1), which play crucial roles in cell proliferation, migration, angiogenesis, and tissue repair. With the development of regenerative medicine and stem cell technology, PL is increasingly being used as a substitute for fetal bovine serum (FBS) for the in vitro expansion of mesenchymal stem cells (MSCs). Studies have shown that human PL has a good effect on supporting MSC proliferation while maintaining their multi-lineage differentiation potential and immunomodulatory properties. Due to its clear source, elimination of animal-derived risks, and promising clinical translation prospects, PL has become an important direction for optimizing cell culture systems.

[0003] Currently, there is a lack of unified standards for PL preparation. Different preparation processes can affect the release efficiency of growth factors and the total protein content. Therefore, it is of great significance to establish a stable and controllable PL preparation system. Summary of the Invention

[0004] The purpose of this invention is to provide a bovine platelet lysate (bPL), its preparation method, and its application. The bovine platelet lysate provided by this invention is rich in various proliferative growth factors, and the composite culture system prepared using it can effectively support the in vitro culture of various cell lines.

[0005] To achieve the above objectives, the present invention provides the following technical solution: This invention provides a bovine platelet lysis buffer, which is obtained by repeatedly freezing and thawing bovine platelets, ultrasonically disrupting them, or using Ga... 2+ It is prepared by pyrolysis through activation.

[0006] This invention also provides a method for preparing bovine platelet lysis buffer, the method comprising: subjecting bovine platelets to repeated freeze-thaw cycles, ultrasonic disruption, or Ga... 2+ The bovine platelet lysate was obtained by activation treatment.

[0007] Preferably, the repeated freeze-thaw cycles are performed by repeatedly freezing and thawing at -80℃ and 37℃ three times, centrifuging at 4000 rpm for 30 min, collecting the supernatant and filtering it through a 0.22μm filter.

[0008] Preferably, the ultrasonic disruption is performed as follows: 285 W ultrasound, 5 seconds of ultrasound followed by a 5-second pause, for a total of ten times; centrifugation at 4000 rpm for 30 minutes, collection of the supernatant and filtration through a 0.22 μm filter.

[0009] Preferably, Ga 2+ Activation is: adding Ga to platelets. 2+ The calcium chloride solution with a final concentration of 4.17 mmol / L was mixed thoroughly, allowed to stand in a water bath for 5 min, centrifuged at 4000 rpm for 30 min, and the supernatant was collected and filtered through a 0.22 μm filter.

[0010] Preferably, the bovine platelet lysate is stored at a temperature below -20°C, and more preferably below -80°C.

[0011] The present invention also provides a composite culture medium containing bovine platelet lysate prepared by the above method.

[0012] Preferably, the compound culture medium is subjected to fibrinogen depletion treatment.

[0013] Preferably, the compound culture medium contains, by weight, 2% ultrasonically disrupted bovine platelet lysate + 8% adult bovine serum, 4% repeatedly freeze-thawed bovine platelet lysate + 6% adult bovine serum, or 4% ultrasonically disrupted bovine platelet lysate + 6% adult bovine serum.

[0014] The present invention also provides the application of the above-mentioned compound culture medium in cell proliferation and cell cloning.

[0015] The present invention also provides a mesenchymal stem cell culture medium, wherein the mesenchymal stem cell culture medium comprises bovine platelet lysate prepared by the above method and DMEM.

[0016] The present invention also provides the application of the above-mentioned mesenchymal stem cell culture medium in adipogenic differentiation or osteogenic differentiation.

[0017] The present invention also provides the application of the above-mentioned mesenchymal stem cell culture medium in supporting cell proliferation in vitro or maintaining multi-directional differentiation capacity. Beneficial effects

[0018] 1. This invention successfully established a method for preparing bPL. Bovine peripheral blood platelets were separated using a double centrifugation method, and then subjected to repeated freeze-thaw cycles, ultrasonic disruption, and Ga... 2+The activation method was used for cleavage, and the effects of different cleavage methods on growth factor release and total protein content were compared. The results showed that all three methods could successfully prepare bPL, with the ultrasonic method showing higher growth factor release, while Ga... 2+ The total protein content is relatively high with the activation method.

[0019] 2. This invention systematically evaluated the effects of different storage temperatures and times on the stability of bPL. The study found that the content of growth factors showed a significant temperature-dependent change with prolonged storage time. Specifically, growth factors maintained good stability within 90 days at -80℃, while some factors decreased at -20℃, and the decrease was more pronounced at 4℃ and room temperature. In contrast, the total protein content changed less, indicating that low-temperature freezing is an important condition for maintaining bPL activity.

[0020] 3. This invention successfully constructed several bPL-based composite culture systems and systematically evaluated the cell proliferation capacity, colony formation capacity, continuous passage stability, and senescence level of different culture systems using six cell models: Vero, ST, MDBK, DF-1, Mode-k, and 293T. Groups F, J, and H (2% sonication + 8% adult bovine serum, 4% freeze-thaw + 6% adult bovine serum, and 4% sonication + 6% adult bovine serum) showed good performance in cell proliferation rate, colony formation capacity, and long-term culture stability, with overall results approaching those of traditional serum culture systems. This is consistent with the optimized bPL preparation results and provides experimental support for the optimization of serum substitution systems.

[0021] 4. This invention further evaluated the effects of the bPL culture system on mesenchymal stem cell culture. The results showed that different culture systems could support stable adherent growth of BM-MSCs and AD-MSCs, maintaining good proliferative capacity and adipogenic and osteogenic differentiation potential. Groups E and F (10% sonication method and 10% Ga²⁺ activation method) showed better or near-perfect performance than the fetal bovine serum culture system in promoting cell proliferation and reducing cell senescence, indicating that bPL has good application potential in the in vitro culture of mesenchymal stem cells, laying the foundation for the application of bPL in MSC culture and regenerative medicine. Attached Figure Description

[0022] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0023] Figure 1The content changes of IGF-1 at different temperatures for 0 days, 30 days, and 90 days are shown. Note: a–d: storage at -80℃, -20℃, 4℃, and room temperature (RT), respectively; bar charts represent freeze-thaw method (black), ultrasonic method (light gray), and Ga²⁺ activation method (dark gray); data are mean ± standard error, n=3, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, ns=no statistical difference.

[0024] Figure 2 The content changes of bFGF at different temperatures at 0d, 30d, and 90d; Note: a–d: storage at -80℃, -20℃, 4℃, and room temperature (RT), respectively; bar charts represent freeze-thaw method (black), ultrasonic method (light gray), and Ga²⁺ activation method (dark gray); data are mean ± standard error, n=3, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, ns=no statistical difference.

[0025] Figure 3 The content changes of PDGF-BB at different temperatures at 0 d, 30 d, and 90 d; Note: a–d: storage at -80℃, -20℃, 4℃, and room temperature (RT), respectively; bar charts represent freeze-thaw method (black), ultrasonic method (light gray), and Ga²⁺ activation method (dark gray); data are mean ± standard error, n=3, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, ns=no statistical difference.

[0026] Figure 4 The content changes of TGF-β1 at different temperatures at 0 d, 30 d, and 90 d; Note: a–d: storage at -80℃, -20℃, 4℃, and room temperature (RT), respectively; bar charts represent freeze-thaw method (black), ultrasonic method (light gray), and Ga²⁺ activation method (dark gray); data are mean ± standard error, n=3, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, ns=no statistical difference.

[0027] Figure 5 The changes in VEGF content at different temperatures at 0, 30, and 90 days are shown. Note: a–d: storage at -80℃, -20℃, 4℃, and room temperature (RT), respectively; bars represent freeze-thaw method (black), ultrasonic method (light gray), and Ga²⁺ activation method (dark gray); data are mean ± standard error, n=3, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, ns=no statistical difference.

[0028] Figure 6Total protein content under different conditions; Note: a–d: storage at -80℃, -20℃, 4℃, and room temperature (RT), respectively; bar charts represent freeze-thaw method (black), ultrasonic method (light gray), and Ga²⁺ activation method (dark gray); data are mean ± standard error, n=3, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, ns=no statistical difference.

[0029] Figure 7 The results are for fibrinogen depletion treatment. Note: FIB+ indicates no fibrinogen depletion was performed; FIB— indicates fibrinogen depletion was performed. ****P<0.0001 indicates statistically significant difference between groups.

[0030] Figure 8 The growth curves of VERO cells under different culture media are shown.

[0031] Figure 9 The growth curves of Mode-K cells under different culture media are shown.

[0032] Figure 10 The growth curves of MDBK cells under different culture media are shown.

[0033] Figure 11 The growth curves of ST cells under different culture media are shown.

[0034] Figure 12 The growth curves of DF-1 cells under different culture media are shown.

[0035] Figure 13 The growth curves of 293T cells under different culture media are shown.

[0036] Figure 14 Cloning results of different cells in various culture media.

[0037] Figure 15 This study compares the cloning results of different cells in various culture media.

[0038] Figure 16 For VERO passage experiments (A: VERO cell count; B: VERO cell viability).

[0039] Figure 17 For Mode-k passage experiments (A: Mode-k cell count; B: Mode-k cell viability).

[0040] Figure 18 For MDBK passage assays (A: MDBK cell count; B: MDBK cell viability).

[0041] Figure 19For ST passage experiments (A: ST cell count; B: ST cell viability).

[0042] Figure 20 For DF-1 passage experiments (A: DF-1 cell count; B: DF-1 cell viability).

[0043] Figure 21 For the 293T passage experiment (A: 293T cell count; B: 293T cell viability).

[0044] Figure 22 To culture to P9 cell morphology; Note: F group: 2% sonication + 8% adult bovine serum; J: 4% freeze-thaw + 6% adult bovine serum; H: 4% sonication + 6% adult bovine serum; G: 10% fetal bovine serum; K: 10% newborn bovine serum; L: 10% adult bovine serum.

[0045] Figure 23 The results represent the senescence of continuously cultured cells above P64; Note: F group: 2% sonication method + 8% adult bovine serum; J: 4% freeze-thaw method + 6% adult bovine serum; H: 4% sonication method + 6% adult bovine serum; G: 10% fetal bovine serum; K: 10% newborn bovine serum; L: 10% adult bovine serum.

[0046] Figure 24 The results are for fibrinogen depletion treatment; Note: FIB+ indicates no fibrinogen depletion was performed; FIB— indicates fibrinogen depletion was performed. ****P<0.0001, the difference between groups is statistically significant.

[0047] Figure 25 Morphology of third-generation AD-MSCs cultured in different culture media; Note: A: 5% freeze-thaw method bPL; B: 5% sonication method bPL; C: 5% Ga²⁺ activation method bPL; D: 10% freeze-thaw method bPL; E: 10% sonication method bPL; F: 10% Ga²⁺ activation method bPL; J: 20% freeze-thaw method bPL; H: 20% sonication method bPL; I: 20% Ga²⁺ activation method bPL; G: 10% FBS.

[0048] Figure 26 Morphology of third-generation BM-MSCs cultured in different culture media; Note: A: 5% freeze-thaw method bPL; B: 5% sonication method bPL; C: 5% Ga²⁺ activation method bPL; D: 10% freeze-thaw method bPL; E: 10% sonication method bPL; F: 10% Ga²⁺ activation method bPL; J: 20% freeze-thaw method bPL; H: 20% sonication method bPL; I: 20% Ga²⁺ activation method bPL; G: 10% FBS.

[0049] Figure 27Adipogenic induction results of 4th generation AD-MSCs cultured in different culture media (Oil Red O staining); Note: A: 5% freeze-thaw method bPL; B: 5% sonication method bPL; C: 5% Ga²⁺ activation method bPL; D: 10% freeze-thaw method bPL; E: 10% sonication method bPL; F: 10% Ga²⁺ activation method bPL; J: 20% freeze-thaw method bPL; H: 20% sonication method bPL; I: 20% Ga²⁺ activation method bPL; G: 10% FBS.

[0050] Figure 28 Adipogenic induction results (Oil Red O staining) of 4th generation BM-MSCs cultured in different culture media; Note: A: 5% freeze-thaw method bPL; B: 5% sonication method bPL; C: 5% Ga²⁺ activation method bPL; D: 10% freeze-thaw method bPL; E: 10% sonication method bPL; F: 10% Ga²⁺ activation method bPL; J: 20% freeze-thaw method bPL; H: 20% sonication method bPL; I: 20% Ga²⁺ activation method bPL; G: 10% FBS.

[0051] Figure 29 Osteogenic induction results of 4th generation AD-MSCs cultured in different culture media (Alizarin Red staining); Note: A: 5% freeze-thaw method bPL; B: 5% sonication method bPL; C: 5% Ga²⁺ activation method bPL; D: 10% freeze-thaw method bPL; E: 10% sonication method bPL; F: 10% Ga²⁺ activation method bPL; J: 20% freeze-thaw method bPL; H: 20% sonication method bPL; I: 20% Ga²⁺ activation method bPL; G: 10% FBS.

[0052] Figure 30 Osteogenic induction results (Alizarin Red staining) of 4th generation BM-MSCs cultured in different culture media; Note: A: 5% freeze-thaw method bPL; B: 5% sonication method bPL; C: 5% Ga²⁺ activation method bPL; D: 10% freeze-thaw method bPL; E: 10% sonication method bPL; F: 10% Ga²⁺ activation method bPL; J: 20% freeze-thaw method bPL; H: 20% sonication method bPL; I: 20% Ga²⁺ activation method bPL; G: 10% FBS Figure 31 Growth curves of 4th generation AD-MSCs cultured in different culture media; Note: A: 5% freeze-thaw method bPL; B: 5% sonication method bPL; C: 5% Ga²⁺ activation method bPL; D: 10% freeze-thaw method bPL; E: 10% sonication method bPL; F: 10% Ga²⁺ activation method bPL; J: 20% freeze-thaw method bPL; H: 20% sonication method bPL; I: 20% Ga²⁺ activation method bPL; G: 10% FBS.

[0053] Figure 32Growth curves of 4th generation BM-MSCs cultured in different culture media; Note: A: 5% freeze-thaw method bPL; B: 5% sonication method bPL; C: 5% Ga²⁺ activation method bPL; D: 10% freeze-thaw method bPL; E: 10% sonication method bPL; F: 10% Ga²⁺ activation method bPL; J: 20% freeze-thaw method bPL; H: 20% sonication method bPL; I: 20% Ga²⁺ activation method bPL; G: 10% FBS.

[0054] Figure 33 Colony formation assays of 5th generation AD-MSCs cells cultured in different culture media; Note: A: 5% freeze-thaw method (bPL); B: 5% sonication method (bPL); C: 5% Ga²⁺ activation method (bPL); D: 10% freeze-thaw method (bPL); E: 10% sonication method (bPL); F: 10% Ga²⁺ activation method (bPL); J: 20% freeze-thaw method (bPL); H: 20% sonication method (bPL); I: 20% Ga²⁺ activation method (bPL); G: 10% FBS.

[0055] Figure 34 The number of clonal clusters of 5th generation AD-MSCs cultured in different culture media; Note: A: 5% freeze-thaw method bPL; B: 5% sonication method bPL; C: 5% Ga²⁺ activation method bPL; D: 10% freeze-thaw method bPL; E: 10% sonication method bPL; F: 10% Ga²⁺ activation method bPL; J: 20% freeze-thaw method bPL; H: 20% sonication method bPL; I: 20% Ga²⁺ activation method bPL; G: 10% FBS.

[0056] Figure 35 Colony formation assays of 5th generation BM-MSCs cells cultured in different culture media; Note: A: 5% freeze-thaw method bPL; B: 5% sonication method bPL; C: 5% Ga²⁺ activation method bPL; D: 10% freeze-thaw method bPL; E: 10% sonication method bPL; F: 10% Ga²⁺ activation method bPL; J: 20% freeze-thaw method bPL; H: 20% sonication method bPL; I: 20% Ga²⁺ activation method bPL; G: 10% FBS.

[0057] Figure 36 Image showing the number of 5th generation BM-MSCs cell clones cultured in different culture media; Note: A: 5% freeze-thaw method bPL; B: 5% sonication method bPL; C: 5% Ga²⁺ activation method bPL; D: 10% freeze-thaw method bPL; E: 10% sonication method bPL; F: 10% Ga²⁺ activation method bPL; J: 20% freeze-thaw method bPL; H: 20% sonication method bPL; I: 20% Ga²⁺ activation method bPL; G: 10% FBS.

[0058] Figure 37Senescence detection of 9th generation AD-MSCs cultured in different culture media; Note: A: 5% freeze-thaw method bPL; B: 5% sonication method bPL; C: 5% Ga²⁺ activation method bPL; D: 10% freeze-thaw method bPL; E: 10% sonication method bPL; F: 10% Ga²⁺ activation method bPL; J: 20% freeze-thaw method bPL; H: 20% sonication method bPL; I: 20% Ga²⁺ activation method bPL; G: 10% FBS.

[0059] Figure 38 Senescence detection of 9th generation BM-MSCs cultured in different culture media; Note: A: 5% freeze-thaw method bPL; B: 5% sonication method bPL; C: 5% Ga²⁺ activation method bPL; D: 10% freeze-thaw method bPL; E: 10% sonication method bPL; F: 10% Ga²⁺ activation method bPL; J: 20% freeze-thaw method bPL; H: 20% sonication method bPL; I: 20% Ga²⁺ activation method bPL; G: 10% FBS. Detailed Implementation

[0060] This invention provides a bovine platelet lysis buffer, its preparation method, and its application. To further illustrate this invention, the technical solution provided by this invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of this invention.

[0061] Unless otherwise specified, the production processes, experimental methods, or testing methods involved in the embodiments of this invention are all conventional methods in the prior art, and their names and / or abbreviations are all conventional names in the field, which are very clear and distinct in the relevant application areas. Those skilled in the art can understand the conventional process steps based on the names and apply the corresponding equipment, and implement them according to conventional conditions or the conditions recommended by the manufacturer.

[0062] The various instruments, equipment, raw materials or reagents used in the embodiments of this invention are not subject to any special restrictions on their source. They are all conventional products that can be purchased through regular commercial channels and can be prepared according to conventional methods known to those skilled in the art.

[0063] This invention uses GraphPad Prism 8.0 software to analyze data. The Student's t-test was used to analyze data between groups. Two-way ANOVA was used to perform significance analysis among multiple groups. Data are expressed as mean ± standard error (mean ± SEM). P < 0.05 is considered statistically significant.

[0064] Example 1: Preparation and quality evaluation of bovine platelet lysis buffer The blood obtained in this embodiment came from eight 25-month-old adult cattle. The sampling site was the teaching animal experimental station of Shihezi University.

[0065] 1.1 Preparation of anticoagulant: Weigh 11 g of sodium citrate, 4 g of citric acid and 12.25 g of glucose and add them to 400 mL of distilled water. Stir to dissolve and add sodium hydroxide solution to make pH=7. Make up to 500 mL. Sterilize and filter through a 0.22 μm filter in a biosafety cabinet and store at 4℃.

[0066] 1.2 Bovine platelet isolation and storage: (1) Bovine blood collection: Sterile blood collection bags were used to ensure that the blood was free from contamination. All the cattle used for blood collection were adult cattle. Blood was collected by jugular vein puncture. 400 mL of blood was collected from each cow, and a total of 8 cows were collected. After the blood collection was completed, the blood was immediately transferred to the biosafety cabinet. Each sample was mixed with anticoagulant and blood at a ratio of 7:50 to prevent blood from agglutinating after collection and affecting platelet separation. If platelets could not be separated in time, the blood containing anticoagulant was stored at 4°C for no more than 24 hours to prevent platelets from spontaneously lysing.

[0067] (2) Blood routine test: Eight samples were subjected to blood routine test to measure the platelet content of each sample.

[0068] (3) Initial separation of bovine platelets: The blood was dispensed into 50 mL centrifuge tubes and centrifuged at 4℃, 2000 rpm for 15 min. After centrifugation, the blood in the centrifuge tubes was observed to be divided into three layers, from top to bottom: plasma layer, white membrane layer, and red blood cell layer. The white membrane layer is mainly composed of white blood cells and platelets.

[0069] (4) Separation of bovine platelets: After removing the centrifuge tube, slowly and gently place it into the biosafety cabinet to prevent the three layers from mixing. Use a syringe to slowly aspirate the middle layer and the plasma layer, avoiding aspirating red blood cells as much as possible. Collect the aspirated solution into a brand new centrifuge tube, using the gentlest possible suctioning motion.

[0070] (5) Final separation of bovine platelets: Balance the new centrifuge tubes and centrifuge at 4°C, 3200 rpm for 20 min. After centrifugation, platelet particles were observed to be deposited at the bottom of the centrifuge tube. Discard the supernatant and keep the precipitate. Add an appropriate amount of physiological saline to each tube and gently resuspend and mix. Collect the platelet mixture from the same group of centrifuge tubes and take samples for routine blood tests.

[0071] Bovine platelet recovery rate: Platelets were obtained by centrifugation under two different conditions, and then the platelet count was obtained by routine blood tests. Platelet recovery rate = total concentrated platelets / total platelets × 100%. The lowest recovery rate was 43.83%, the highest was 61.92%, the difference was 18.09%, and the average recovery rate was 50.10% ± 5.73% (Table 1).

[0072] Table 1. Bovine platelet recovery rate ; 1.3 Preparation of bovine platelet lysis buffer: (1) bPL dispensing: Transfer the platelet mixture to a biosafety cabinet and divide it into three equal portions labeled A, B and C.

[0073] (2) Group A freeze-thaw treatment: Repeated freeze-thaw at -80℃ and 37℃ three times, centrifuged at 4000 rpm for 30 min, collected the supernatant and filtered with a 0.22μm filter.

[0074] (3) Group B ultrasonic treatment: 285 W ultrasonication (5 s for 5 s, 5 s for 5 s, 10 times in total). Centrifuge at 4000 rpm for 30 min, collect the supernatant and filter it with a 0.22 μm filter.

[0075] (4) Calcium chloride activation in group C: 200 μL of 0.025 mol / L calcium chloride solution (Ga) was added to platelets. 2+ The final concentration was 4.17 mmol / L. After mixing thoroughly, the mixture was allowed to stand in a water bath for 5 min. The mixture was then centrifuged at 4000 rpm for 30 min, and the supernatant was collected and filtered through a 0.22 μm filter.

[0076] (5) bPL storage: Seven samples were taken from each group, one for platelet content detection and the remaining six for growth factor content detection. Groups A, B and C were all stored in a -80 ℃ refrigerator to ensure their stability.

[0077] (6) Blood routine test: Blood routine tests were performed on groups A, B and C respectively to determine the platelet content to determine whether the platelets had lysed completely. If the lysis was complete, the operation was repeated.

[0078] From the perspective of residual platelet count, i.e., the highest lysis efficiency, the overall performance of the three methods is ranked as follows: Group B (ultrasound method), Group C (Ga²⁺ activation), and Group A (freeze-thaw method). Group B (ultrasound method) had the lowest total platelet count after two treatments, at only 8 × 10⁻⁶. 9 Cell / L, accounting for 4.6% of the initial total. This indicates that ultrasound, under the parameters in Method 2.1.6, achieves the most thorough physical disruption of platelets, minimizing the residue of intact platelets. In Group C (Ga²⁺ activated), the total platelet count decreased to 17 × 10⁶ after only one treatment. 9 The cell count was 9.8% of the initial total. This method achieves a high lysis rate through chemical activation alone, and no secondary treatment was performed to prevent excessively high Ga²⁺ concentration from affecting subsequent cell culture. Even after secondary treatment, the total remaining platelet count in Group A (freeze-thaw method) was still 21 × 10⁻⁶. 9Cell / L, accounting for 12.1% of the initial total, is the highest residual amount among the three. This indicates that after three freeze-thaw cycles, it is not sufficient to completely lyse platelets, but if stored and used repeatedly afterward, platelets will continue to lyse.

[0079] Table 2 Preparation of bovine platelet lysate

[0080] 1.4 Detection of growth factor content (1) The three groups of samples A, B and C were stored at room temperature, 4℃, -20℃ and -80℃ for 0 days, 30 days and 90 days respectively. The contents of total protein, bFGF, TGF-β1, PDGF-BB, VEGF and IGF-1 were detected.

[0081] (2) Reagent preparation and sample addition: Take out each component of the ELISA kit and equilibrate at room temperature for 20 min. Set up standard wells, sample wells and blank wells in sequence on the microplate; add 50 μL of standard of a series of concentrations to the standard wells; add 10 μL of bovine platelet lysis buffer to the sample wells first, and then add 40 μL of sample diluent; do not add any sample to the blank wells.

[0082] (3) Incubation with primary antibody: Add 100 μL of HRP-labeled detection antibody to each well, except for the blank wells. Cover with sealing film and incubate at 37℃ for 60 min.

[0083] (4) Washing: After incubation, discard the liquid in the well and pat dry on absorbent paper. Fill each well with washing solution, let stand for 30 seconds and then discard. Repeat this process 5 times, and finally pat dry.

[0084] (5) Colorimetric reaction: Add 50 μL of substrate A and substrate B to each well sequentially, and gently shake to mix. Cover with a new sealing film and react at 37°C in the dark for 15 min. (6) Termination of assay: Add 50 μL of stop solution to each well and mix gently to terminate the reaction. Within 15 min after the reaction is terminated, measure the absorbance (OD) value of each well at 450 nm using a microplate reader. (7) Calculation results: Based on the concentration of the standard wells and the corresponding OD values, a standard curve (working curve) was plotted. The OD values ​​of the sample wells were substituted into the curve equation to calculate the content of the target cytokines in the bovine platelet lysate sample. This invention compares three preparation methods (freeze-thaw method, ultrasonic method, and Ga) 2+The effects of activation method on the release levels and stability of five growth factors were investigated. To systematically evaluate the effects of different storage conditions on the stability of multiple growth factors, a comprehensive analysis was conducted on the changes in the content of IGF-1, bFGF, PDGF-BB, TGF-β1, and VEGF after storage at −80℃, −20℃, 4℃, and room temperature (RT) for 0, 30, and 90 days. The results showed that… Figure 1-5 The release levels of the five growth factors differed at 0 days using three preparation methods (freeze-thaw method, ultrasonic method, and Ga²⁺ activation method). The ultrasonic and freeze-thaw groups showed relatively higher initial release levels of IGF-1, bFGF, and PDGF-BB. The calcium ion activation group showed relatively more stable initial concentrations of some factors (such as TGF-β1), and the differences among the three groups for some growth factors were statistically significant (P < 0.05). All five growth factors exhibited obvious temperature- and time-dependent trends. The specific changes in content at different temperatures (−80℃, −20℃, 4℃, room temperature) and storage times (0 days, 30 days, 90 days) are as follows: At −80℃, each factor maintained high stability overall within 90 days, with most time points showing no statistically significant differences or only slightly significant differences, and no progressive significant decrease. At −20℃, each factor gradually decreased with prolonged storage time; at 30 days, some comparisons showed no significant differences, while at 90 days, many factors decreased significantly. At 4℃, all growth factors showed a significant decrease after 30 days, with significant differences observed among multiple groups. The decrease intensified further at 90 days, exhibiting a clear cumulative effect over time. At room temperature, the five growth factors were most significantly affected, showing a highly significant decrease after 30 days, and reaching low levels at 90 days, with some factors approaching the low detection range. The stability of the five growth factors was ranked as follows: −80℃ > −20℃ > 4℃ > Room temperature.

[0085] 1.5 Total Protein Content Detection (1) Preparation of working solution: According to the kit instructions, mix reagent 1 and reagent 2 at a volume ratio of 1:2 to prepare biuret working solution for later use.

[0086] (2) Sample addition reaction: Take a clean 5 mL centrifuge tube, add 50 μL of the bPL sample to be tested and 2.5 mL of biuret working solution in sequence, and vortex to mix.

[0087] (3) Incubation and color development: Place the mixture in a 37°C constant temperature water bath or incubator and incubate in the dark for 10 min.

[0088] (4) Cooling: After incubation, immediately place the centrifuge tube under running tap water to cool to room temperature to terminate the reaction.

[0089] (5) Absorbance measurement: Using a UV-Vis spectrophotometer, with double-distilled water as a blank control for zeroing, the absorbance value of the reaction solution was measured at a wavelength of 540 nm and a light path of 1 cm.

[0090] (6) Concentration calculation: Based on the measured absorbance value, substitute it into the pre-plotted standard curve equation or the calculation formula provided by the kit to calculate the total protein concentration in the sample.

[0091] This invention uses the BCA method to detect changes in total protein content of platelet lysates prepared by different methods after storage at -80℃, -20℃, 4℃, and room temperature for 0 days, 30 days, and 90 days. The results show ( Figure 6 Under all storage temperatures and time points, statistically significant differences were observed in the total protein content among different preparation methods. The overall ranking of total protein content among different preparation methods was: Ga2+ activation method first, freeze-thaw method second, and ultrasonic method third. At -80℃ and -20℃, the differences in total protein content among the three preparation methods at each time point (0 d, 30 d, 90 d) were statistically significant (P < 0.05 or P < 0.01), and under the same preparation method, the total protein content did not change significantly with prolonged storage time (P > 0.05). At 4℃, the total protein content of each group showed a slight decreasing trend with prolonged storage time, and significant differences remained between different preparation methods at each time point. At room temperature, the total protein content showed a decreasing trend with prolonged storage time, with the freeze-thaw method and ultrasonic method showing more significant decreases; Ga2+ activation method first, freeze-thaw method second, and ultrasonic method third. 2+ The activation group also showed a downward trend, but the overall level of total protein content was still higher than that of the other two groups.

[0092] Example 2: Preparation and efficacy verification of compound formulation culture medium 2.1 Preparation of compound culture medium The culture medium formula used in this embodiment is detailed in Table 3. After preparation, it should be stored at 4°C for no more than 7 days.

[0093] Table 3 Preparation of composite culture medium ; 2.2 Fibrinogen Detection Equilibrate all reagents to room temperature. Set up standard wells (50 μL of serially concentrated standards), sample wells (10 μL of sample and 40 μL of diluent), and blank wells on the microplate. Add 100 μL of HRP-labeled antibody to each well and incubate at 37 °C for 60 min. After incubation, discard the solution and wash five times. Pat dry and add 50 μL each of substrates A and B sequentially. Incubate at 37 °C in the dark for 15 min. Finally, add stop solution to each well and measure the OD value at 450 nm using a microplate reader within 15 min. Plot a standard curve based on the standard well data and calculate the content of the target cytokine in the sample using the curve equation.

[0094] 2.3 Fibrinogen Depletion Treatment After preparing the culture medium according to method 2.1, store the medium at 4°C overnight. The next day, incubate it in a water bath at 37°C for 2 hours, and fibrin clots can be observed. Remove the medium and transfer it to a biosafety cabinet. Use a pipette tip to break up the fibrin clots, centrifuge at 4000 rpm for 30 min at room temperature, collect the supernatant, filter it aseptically with 0.22 μm filter, and store it at 4°C for later use.

[0095] like Figure 7 As shown, all experimental groups containing bPL exhibited agglutination after overnight incubation at 4°C, while the control group did not. The experimental groups underwent fibrinogen depletion treatment, and the fibrinogen content in the culture medium before and after the treatment was detected by EIIiSA. After treatment, the fibrinogen content in groups A, B, C, D, F, and I was lower than the minimum detection value, calculated as 0.1% of the minimum value. Calculated at μg / mL, the fibrinogen levels in the nine treatment groups decreased by 98.05% (P < 0.0001), 98.39% (P < 0.0001), 98.60% (P < 0.0001), 98.77% (P < 0.0001), 98.71% (P < 0.0001), 95.50% (P < 0.0001), 92.88% (P < 0.0001), 92.61% (P < 0.0001), and 97.57% (P < 0.0001), respectively. The fibrinogen content in all experimental groups was significantly reduced (P < 0.0001), with removal rates ranging from 92.61% to 98.77%. These results indicate that the depletion treatment can efficiently remove fibrinogen from bPL medium, thereby resolving the agglutination problem it causes.

[0096] 2.4 Cell resuscitation and passage Remove the cell cryovials from the liquid nitrogen tank and immediately place them in a 37°C water bath. Gently shake to thaw completely within 1 minute. Wipe the outside of the cryovials with a 75% ethanol cotton ball and transfer them to a biosafety cabinet. Transfer the cell suspension to a 15 mL centrifuge tube containing 2 mL of complete culture medium. Centrifuge at 1000 rpm for 5 minutes, discard the supernatant, add 5 mL of fresh complete culture medium, gently resuspend the cell pellet, transfer it to a T25 flask, and incubate at 37°C with 5% CO2 for 24 hours. After observing that the cell density reaches 80-90% confluence, the culture medium in the original culture flask is aspirated, an appropriate amount of 3 mL PBS is added, and after gentle shaking, the residual serum is discarded. An appropriate amount of 1 mL trypsin is added, and the mixture is gently shaken to cover all cells with the digestive solution. The flask is then placed in a 37℃ incubator for 1-2 min. Under a microscope, once the intercellular spaces have widened and the cells have become more rounded, 2 mL of complete culture medium is added to stop the digestion. The cells are then gently pipetted to the bottom of the flask several times to completely detach them. The cell suspension is transferred to a 15 mL centrifuge tube, centrifuged at 1000 rpm for 5 min, the supernatant is discarded, an appropriate amount of complete culture medium is added, and the cells are gently pipetted to prepare a single-cell suspension, which is then added to a T25 flask containing 4 mL of complete culture medium.

[0097] 2.5 Cell Culture Experiment Vero, Mode-k, MDBK, ST, DF-1, and 293T cells, which have been passaged for three or more consecutive generations, were seeded into 96-well plates at a cell density of 1 × 10⁶ cells per well. 4 Cells were cultured at 0.2 mL / well at 37 °C and 5% CO2 for 7 days. CCK-8 reagent was added to each well at a ratio of 10% of the total culture medium volume and mixed thoroughly to prepare the CCK-8 detection working solution. Each day, the cell culture plate was removed, the original culture medium was discarded, and the cells were washed with PBS. CCK-8 detection solution was added to each well, and the plates were incubated under the same conditions for 2 hours. Subsequently, the optical density of each well was measured at 450 nm using a microplate reader to plot the cell growth curve.

[0098] The results are as follows Figure 8-13As shown, Vero, Mode-k, MDBK, ST, DF-1, and 293T cells were cultured using twelve different culture media. After 7 days of continuous culture, OD values ​​were measured to create growth curves. All cell lines followed the classic S-shaped growth curve under different treatments, experiencing a latent phase, logarithmic growth phase, and plateau phase. Almost all cells were in a slow growth phase from 24 to 72 hours, gradually entering the logarithmic growth phase after 96 hours, and reaching a peak proliferation rate at 144 to 168 hours. However, different serum formulations significantly altered the morphological characteristics of the curves. In Vero cells, the overall ranking of the endpoint OD450 values ​​was as follows: group F had the highest value, followed by groups H, E, and G; groups J and K were in the middle; group L was slightly lower; and groups A, B, C, and D were relatively weaker. In ST cells, group F showed the strongest performance, followed by groups H and E; groups I and J were at a moderate level; groups G were relatively close to groups K and L but slightly lower than the dominant groups; and groups A, B, and C had the lowest proliferation capacity. In Mode-k cells, group J showed the highest performance, followed by groups F, I, and H. Groups G, K, and L were at a mid-to-high level, while the other treatment groups were lower. The ranking of MDBK cells was similar to Mode-k, with group J being the best, followed by groups F and H. Group G was at a mid-level, and groups K and L were slightly lower. In DF-1 cells, group H showed the most significant proliferation effect, followed by group E. Groups B and J were at a mid-to-high level, while groups G, K, and L were generally similar but lower than groups H and E. The overall trend of 293T cells was similar to VERO, with groups F and H being dominant, and group G at a mid-level. Internal comparisons of the three control groups (G, K, L) showed that group G had a slightly higher endpoint OD value in most cells than groups K and L, and the growth trend was more stable; group K showed fluctuations at some time points, and group L was slightly lower overall. Therefore, group G can be considered the best performing control group among the three. Subsequent comparisons between the dominant experimental groups F, J, and H and group G revealed the following: In VERO, ST, and 293T cells, group F showed a significantly higher endpoint OD value and a steeper proliferation curve slope than group G; in Mode-k and MDBK cells, group J outperformed group G; and in DF-1 cells, group H significantly outperformed group G. Group F maintained medium-to-high proliferation levels in all six cell types without showing a significant inhibitory trend compared to group G, while groups J and H exhibited some cell type selectivity.

[0099] 2.6 Cell Cloning Experiment According to the results in section 2.5, Vero, Mode-k, MDBK, ST, DF-1, and 293T cells were cultured in the medium with the growth curve closest to that of the fetal bovine serum group. Vero, Mode-k, MDBK, ST, DF-1, and 293T cells were seeded into 6-well plates at a density of 0.5 × 10³–1 × 10³ cells / mL, 2 mL / well. The plates were incubated at 37 ℃ and 5% CO₂ for 7–14 days. After incubation, the cells were washed twice with PBS, and 1 mL of 4% paraformaldehyde was added to each well for fixation at room temperature for 15 min. The paraformaldehyde was discarded, and the cells were washed twice with PBS. 1 mL of 0.1% crystal violet was added to each well for staining at room temperature for 15 min. The crystal violet was discarded, and the cells were rinsed thoroughly with running water and then air-dried upside down.

[0100] The results are as follows Figure 14-15 As shown, different cell types exhibit significant differences in their response to the culture system, but the overall trend follows a certain regularity. Vero cells showed significant differences under different culture conditions. Group G culture medium produced the most clones with the largest and densest distribution, demonstrating the strongest colony-forming ability; Group F was second; Group K showed a significant decrease; and Group L was the weakest, with sparse and small clones. The overall ranking was: G>F>K>L. Mode-k cells were relatively sensitive to the culture system, with Group G producing the most clones with the most uniform distribution; Group K was second; Group F was lower; and Group L was the lowest. The ranking was: G>K>F>L. MDBK cells formed the most dense and largest clones under Group G conditions; Group F was second; Group K further decreased; and Group L was the weakest. The ranking was: G>F>K>L. ST cells formed a large number of well-defined clones under Group G culture medium, showing the best performance; Group F was slightly lower; and Groups K and L decreased in that order. The ranking was: J>F>K>L. DF-1 cells formed the densest and largest colonies in group H culture medium, exhibiting the strongest colony-forming ability; group G was second; group K was next; and group L was relatively lower. The overall ranking was: H>J>K>L. 293T cells formed the most and most evenly distributed colonies in group F culture medium, showing the best performance; group G was second; group K was next; and group L was the lowest. The ranking was: F>G>K>L.

[0101] 2.7 Cell passage experiments According to the results in 2.5, Vero, Mode-k, MDBK, ST, DF-1, and 293T cells were cultured in the culture medium with the growth curve closest to that of the fetal bovine serum group. 1 mL of 0.25% trypsin was added to T25 culture medium to digest the cells, followed by the addition of 2 mL of complete culture medium to terminate the digestion. The cell suspension was collected, centrifuged at 1000 rpm for 5 min, the supernatant was discarded, and the cells were resuspended in PBS to prepare a cell suspension. Samples were taken, and trypan blue staining was added. Cell count and viability were determined using an automated cell counter. The cell number was 0.7 × 10⁶ cells / mL. The above procedure was repeated every two days for five consecutive passages.

[0102] like Figure 16-21 As shown, after continuous culture to the 7th generation, the cell number and viability of the six cell lines in different culture systems (F group, G group, K group, L group and corresponding optimal group) were detected. The results showed that all experimental culture media could support the stable expansion of the six cell lines: Vero, Mode-k, MDBK, ST, DF-1, and 293T. The cell number showed a regular increasing trend with each passage, and no obvious proliferation arrest or growth decline was observed between generations. The overall proliferation curves of each experimental culture media group were basically consistent with those of the FBS group (fetal bovine serum group), and no obvious growth delay or decreased expansion capacity was observed. The selected superior culture media could maintain the continuous proliferation capacity of the cells. Cell viability detection results showed that the viability of each cell line remained at a high level during continuous passage, without a significant decreasing trend with increasing passages. Furthermore, the difference in viability between the experimental groups and the FBS group was small, and the overall viability remained within a stable range.

[0103] After continuous culturing to the 7th generation, the morphological state of the six cell types was observed under a microscope in different culture systems (F group, G group, K group, L group, and the corresponding optimal group). The results showed ( Figure 22The growth density, adhesion, and morphological integrity of different cells varied under different culture conditions. Vero cells all exhibited typical epithelial-like cell morphology: clear granules, distinct boundaries, and tightly packed cells forming a "paving stone"-like monolayer structure; the overall cell morphology was robust, and there were no significant differences in morphological characteristics among the groups. Mode-k cells also exhibited typical epithelial-like cell morphology, with cells predominantly short spindle-shaped or elliptical, growing dispersedly with clear intercellular spaces; individual cells had distinct outlines and visible granules; cells adhered firmly to the wall, exhibiting robust overall morphology and good cell membrane refractive properties; there were no significant differences in morphological characteristics among the groups. MDBK cells were predominantly polygonal or short spindle-shaped; in a dispersed growth state, the cytoplasm was convex, the boundaries were clear, and there was no intracellular granule accumulation; the cells had strong adhesion ability, and there were no significant differences in morphological characteristics among the different groups of MDBK cells. ST cells are predominantly spindle-shaped, exhibiting typical island-like colony growth. At low densities, the cell colony edges are clear, the cell bodies are translucent, and there are no obvious intracellular granules. Cells adhere firmly to the culture wall, and the island-like growth morphology of ST cells from different passages and culture conditions remains consistent. DF-1 cells exhibit a typical fibroblast-like morphology, mostly slender spindle-shaped, with clearly extended cell bodies and interwoven structures forming a network. Adhesion is good, cell density is high, and there are no significant abnormalities in morphological characteristics among the groups. 293T cells exhibit polygonal or epithelial-like adherent growth, with clear cell boundaries, plump cytoplasm, and uniform arrangement forming a continuous monolayer. Cell junctions are tight, overall growth is good, and morphological characteristics are basically consistent across groups, with no significant morphological differences observed.

[0104] 2.8 Cell senescence experiment Six cell lines (Vero, Mode-k, MDBK, ST, DF-1, and 293T) were cultured continuously from P58 to P64 passages, then passaged into 24-well plates and cultured for 24 h. After washing with PBS, 250 μl of β-galactosidase staining solution was added to fix the cells at room temperature for 15 min. After removing the cell fixative, the cells were washed with PBS at least three times for 3 min each time. 250 μl of β-galactosidase staining working solution was added to each well. The plates were sealed with film to prevent CO2 from entering and incubated overnight at 37°C.

[0105] Preparation of working solution for β-galactosidase staining: 60 μL of β-galactosidase staining solution A, 60 μL of β-galactosidase staining solution B, 55-80 μL of β-galactosidase staining solution C, and 300 μL of X-Gal solution.

[0106] like Figure 23As shown, this embodiment uses senescence-associated β-galactosidase (SA-β-gal) staining to detect six cell types after continuous passage to the 64th generation in F, G, K, and L cell culture systems. Blue-green deposits indicate positive cells, suggesting that different culture systems have varying effects on the senescence level of the six cell types, but the overall trend is relatively consistent. In Vero, Mode-k, MDBK, and ST cells, the proportion of positive cells in group G was the lowest or near the lowest, followed by group F, with group K showing a further increase. Group L had the highest number of blue-stained positive cells and the deepest staining intensity, indicating the most significant senescence. In DF-1 cells, group H had the lowest positive rate, group G was slightly higher, and groups K and L increased sequentially. In 293T cells, group F had the lowest senescence level, followed by groups G and K, with group L having the highest. In summary, group L showed a high SA-β-gal positive rate in all cell types, group G maintained a low senescence level in most cell types, and group F had a certain advantage in some cell types (especially 293T).

[0107] 2.9 Summary (1) Successfully constructed a variety of compound culture medium systems and completed fibrinogen depletion treatment and related quality control to make them have stable and reproducible in vitro cell culture application conditions.

[0108] (2) Using six cell types, namely Vero, ST, MDBK, DF-1, Mode-k and 293T, as models, we systematically carried out functional evaluations such as growth curve determination, continuous passage observation, cell colony formation experiment and β-galactosidase senescence detection, and established a multi-index comprehensive evaluation system.

[0109] (3) Through comparison within the control group and systematic screening between experimental groups, the compatibility differences of different compound formulations in different cells were clarified. Finally, the F, J, and H groups (2% sonication method + 8% adult bovine serum, 4% freeze-thaw method + 6% adult bovine serum, and 4% sonication method + 6% adult bovine serum) were selected as superior culture media with stable proliferation, strong cloning ability and low senescence level in the cell model, providing experimental basis for subsequent optimization of serum replacement system.

[0110] Example 3: Effects of culturing mesenchymal stem cells with bovine platelet lysate. The feline adipose-derived stem cells (AD-MSCs) and feline bone marrow mesenchymal stem cells (BM-MSCs) used in this embodiment were isolated by the Basic Laboratory of Shihezi University.

[0111] 3.1 Culture medium preparation The culture medium formula used in this embodiment is detailed in Table 4. After preparation, it should be stored at 4°C for no more than 7 days.

[0112] Table 4. Culture medium preparation ; 3.2 Reagent Preparation Dexamethasone Concentrate A: Add 250 mg of dexamethasone to a 50 mL centrifuge tube, dilute to volume with 25 mL of anhydrous ethanol to prepare a 2.5 mM concentrate. Filter at 0.22 μm and dispense into 4 mL tubes. Store at -20°C protected from light.

[0113] IBMX concentrate: Weigh 100 mg IBMX and add it to a 1 mL EP tube, then add 900 μL DMSO to prepare a 0.5 M concentrate. Store frozen at 20°C.

[0114] Preparation of adipogenic differentiation induction medium I: Take a 50 mL centrifuge tube and add 10 μL of dexamethasone concentrate A, 25 μL of IBMX, 71.5 μL of insulin, 2.5 mL of FBS, and 250 μL of penicillin-dextrose antibody. Adjust the volume to 25 mL with DMEM medium. The final concentration is 1 μL dexamethasone, 0.5 mM IBMX, 10 μg / mL insulin, and 10% FBS.

[0115] Preparation of adipogenic differentiation induction culture medium II: Add 71.5 μL insulin, 2.5 mL FBS, and 250 μL penicillin antibody to the container, and bring the volume to 25 mL with DMEM medium. The final concentration is 10 μg / mL insulin and 10% FBS. It should be prepared fresh for use.

[0116] Preparation of Oil Red O staining solution: This staining solution is prepared fresh for use. Mix Oil Red O staining A and Oil Red O staining B in a 3:2 ratio, filter through a 0.22 μm filter, and let stand in the dark for 10 min.

[0117] Dexamethasone Concentrate B: Add 0.1 g of dexamethasone to a 50 mL centrifuge tube and dilute to volume with 25 mL of anhydrous ethanol to prepare a 10 mM concentrate. Filter at 0.22 μm and dispense into 4 mL tubes. Store at -20℃ protected from light.

[0118] Osteogenic differentiation induction concentrate: Take 50 mL centrifuge tubes and add 0.025 g of L-ascorbic acid, 1.5306 g of sodium glycerophosphate, and 5 μL of dexamethasone concentrate B. Adjust the volume to 25 mL with DMEM medium. The final concentration is 50 μg / mL ascorbic acid, 10 mM sodium glycerophosphate, and 100 mM dexamethasone. Aliquot into 2.5 mL tubes.

[0119] Osteogenic differentiation induction medium: 2.5 mL, osteogenic differentiation induction concentrate 5 mL FBS, 42 mL DMEM, 500 μL double antibiotics.

[0120] 3.3 Fibrinogen Depletion Treatment Similar to method 2.3, the culture medium was stored overnight at 4°C. The next day, it was incubated in a water bath at 37°C for 2 hours, and fibrin clots were observed. The medium was then removed and transferred to a biosafety cabinet. The fibrin clots were broken up using a pipette tip and centrifuged at 4000 rpm for 30 minutes at room temperature. The supernatant was collected, aseptically filtered through a 0.22 μm filter, and stored at 4°C for later use.

[0121] The changes in fibrin concentration before and after depletion were detected using three treatment methods (freeze-thaw A, ultrasound B, and calcium ion C) at three concentrations (5%, 10%, and 20%). After treatment, the concentrations in each group decreased by 80.45% (P < 0.0001), 80.82% (P < 0.0001), 75.23% (P < 0.0001), 82.03% (P < 0.0001), 82.27% (P < 0.0001), 75.48% (P < 0.0001), 74.02% (P < 0.0001), 71.74% (P < 0.0001), and 62.01% (P < 0.0001), respectively. Figure 24 Data showed that the fibrin concentration decreased significantly in all treatment groups after depletion, proving that this method effectively consumed fibrin. The calcium ion C group had higher post-depletion concentrations at all concentrations than the freeze-thaw A and ultrasonic B groups at the same concentrations, suggesting that calcium ion treatment may have a relatively weaker effect on fibrin consumption. Higher concentrations resulted in higher residual concentrations after depletion, with the 20% concentration group showing the highest residual calcium ion concentrations after all treatments (mean 3.3089 µg / mL). Freeze-thaw A and ultrasonic B treatments showed higher consumption efficiency at lower concentrations, while calcium ion C showed higher residual levels at all concentrations.

[0122] 3.4 Resuscitation and Culture of BM-MSCs and AD-MSCs Preheat 10% complete culture medium, trypsin, and PBS in a 37°C water bath for 40 min. Remove the cell cryopreservation tubes from the liquid nitrogen tank, thaw and sterilize them rapidly at 37°C, and then transfer them to a biosafety cabinet. Transfer the cell suspension to centrifuge tubes containing culture medium, centrifuge, discard the supernatant, resuspend the cell pellet in culture medium, add it to a T25 flask, and incubate at 37°C with 5% CO2 for 24 h. Subculture using the 10 culture media listed in Table 4 according to normal procedures.

[0123] Morphological observation of BM-MSCs and AD-MSCs: Observation of cell morphological changes using an inverted microscope showed that MSCs from both sources could adhere well and grow in various culture media. The cell morphology was mainly long spindle-shaped or fusiform, with some cells arranged in whorls or parallel bundles. The overall morphology was consistent with the typical morphological characteristics of mesenchymal stem cells. Figure 25 , Figure 26 ).

[0124] Under different culture medium conditions, the growth density and spreading degree of the two cell types differed to some extent. Both AD-MSCs and BM-MSCs were supported for adherent growth in the composite culture medium containing bPL. In some medium groups, cells spread sufficiently, arranged relatively regularly, and had high cell density; while in others, cell density was relatively low, and intercellular spacing was larger, but the overall morphology still maintained typical MSC-like characteristics. In the control group G (containing 10% fetal bovine serum), both cell types showed good adhesion and high cell density, with uniform cell morphology and a typical fibroblast-like arrangement. Comparison of the morphological characteristics of the two cell sources revealed that AD-MSCs were relatively slender overall, with some areas showing a clear tendency for parallel arrangement; BM-MSCs, on the other hand, exhibited more uniform spindle-shaped or short spindle-shaped cells, with a relatively large cell spreading area and tighter intercellular connections. Under most culture medium conditions, both cell types maintained stable adherent growth, and no obvious cell rounding or detachment was observed, indicating that the constructed composite culture medium system can support the basic growth requirements of MSCs from both sources in vitro.

[0125] 3.5 Identification of adipogenic differentiation of BM-MSCs and AD-MSCs Take 3rd generation cells cultured at 3.4°C and store them at 5 × 10⁶ cells per well. 4Cells were seeded at a density suitable for both experimental (AI) and control (G) culture media in 6-well plates. Each well was incubated at 37°C with 5% CO2 saturated humidity. The medium was changed every two days, with the corresponding medium added. Cell proliferation was observed using an inverted microscope. When cell confluence reached 100%, a 48-hour contact inhibition treatment was initiated to synchronize cell growth. Simultaneously, the adipogenic differentiation induction medium I was removed and brought to room temperature in the dark. The old medium was discarded, and the cells were washed with 37°C PBS, gently aspirated three times to prevent cell curling. Adipogenic differentiation induction medium I was added and cultured for 3 days. The medium color and cell status were observed daily. If the medium turned yellow, adipogenic differentiation induction medium I was replaced. After 3 days, adipogenic differentiation induction medium II was brought to room temperature in the dark, adipogenic differentiation induction medium I was discarded, and adipogenic differentiation induction medium II was slowly added and cultured for 4 days. The same procedure was repeated, observing the medium color and cell status daily. If the medium color turned yellow, adipogenic differentiation induction medium II was replaced. After d, discard the lipid-inducing differentiation culture medium II and add complete culture medium containing 10% FES for culture. Observe the cell status under a microscope. When 70% of the lipid droplets have precipitated, perform staining treatment.

[0126] Staining procedure: Discard the old culture medium, wash with 37℃ PBS, gently aspirate three times, add 1 mL of 4% paraformaldehyde fixative to cover the bottom of the well, and fix at room temperature for 20-30 min. Discard the fixative, wash twice with PBS, add 1 mL of prepared Oil Red O staining solution, stain at 37℃ in the dark for 60 min, and observe under a microscope.

[0127] like Figure 27-28 As shown, in the adipogenic induction results of AD-MSCs, cells cultured in various media all showed varying degrees of lipid droplet deposition, but the staining intensity and lipid droplet distribution differed. Groups A, B, and C showed a large number of red lipid droplets, with some areas exhibiting clustered distribution and more pronounced staining, indicating good adipogenic differentiation ability. Lipid droplet formation was also observed in groups D and E, but the overall number was slightly less and the distribution was relatively dispersed. Lipid droplet deposition was relatively limited in groups F, G, and H, with only scattered red granules appearing in localized areas and showing weaker staining intensity. A certain number of lipid droplets were observed in groups I and J, but the overall staining density was lower than that of groups A–C.

[0128] In the adipogenic induction results of BM-MSCs, Oil Red O-positive lipid droplet deposition was also observed in the cells, but the differences between different culture media were quite significant. Groups A, B, and C showed relatively abundant lipid droplet deposition, with large staining areas and lipid droplets mostly distributed in clusters or sheets, indicating strong adipogenic differentiation capacity. Groups D and E also showed obvious lipid droplet staining, but the overall density was slightly lower than that of groups A–C. Groups F and H showed a certain number of lipid droplets, but their distribution was relatively scattered, and the staining degree was relatively weak. Groups G and I had fewer lipid droplets, with only localized scattered deposition. Group J also showed some degree of lipid droplet formation, but the overall staining area and lipid droplet density were lower than the dominant groups.

[0129] 3.6 Identification of osteogenic induction differentiation of BM-MSCs and AD-MSCs Fourth-generation cells cultured at 3.4°C were used at a density of 5 × 10⁶ cells per well. 4 Cells were seeded at a density suitable for osteogenic differentiation induction medium (AI) in one well and 2 mL of the control group's G medium in another well. The cells were incubated at 37°C with 5% CO2 and saturated humidity. Microscopic observation was performed. When the cell confluence reached 80%, the medium was replaced with osteogenic differentiation induction medium. The medium was changed every two days. Cell status was observed; if the culture color turned yellow, it indicated that nutrients were depleted and the medium needed to be replaced earlier. Staining was performed after 14 days of continuous culture.

[0130] Staining procedure: Carefully aspirate the culture medium, add 1 mL of Ob fixative to each well and fix for 15 min, add 1 mL of PBS to each well and wash 3 times for 3 min each time, add alizarin red S staining solution and wash for 30 min, wash off the staining solution with distilled water and observe under a microscope.

[0131] like Figures 29-30 As shown, after osteogenic induction culture, the cell morphology of AD-MSCs gradually changed from the original spindle-shaped fibrous structure to polygonal or radial arrangement, with increasingly tighter intercellular connections and increased extracellular matrix deposition. Alizarin red staining results showed that different degrees of red or dark red mineralized nodules were observed in all culture media groups, indicating that AD-MSCs possessed certain osteogenic differentiation potential under different culture systems. There were certain differences in the degree of mineralization among different culture media. Groups B, E, and H showed more obvious staining, with a larger number and denser distribution of mineralized nodules. Groups C, D, and I also showed relatively obvious mineralization deposition, but the overall staining degree was slightly weaker. Groups A, F, and J had relatively fewer mineralized nodules and a more dispersed distribution. The control group G (10% FBS medium) also formed a certain number of mineralized nodules, with its overall mineralization level at a moderate level compared to the experimental groups.

[0132] BM-MSCs also exhibited significant morphological changes during osteogenic induction culture. With prolonged induction culture, the cells gradually transformed from a fibrous morphology to a polygonal or stellate arrangement, accompanied by a gradual increase in extracellular matrix. Alizarin Red staining results showed varying degrees of calcium salt deposition in all groups, forming red mineralized nodules of different sizes. Intergroup comparisons revealed that groups B, E, and H had a greater number of mineralized nodules with a wider staining range, exhibiting more pronounced red deposition; groups C, D, and I also formed a certain number of mineralized nodules, but the overall staining intensity was relatively weak; while groups A, F, and J had less mineralization and the nodules were more dispersed. The control group G also showed significant mineralized nodule formation, with its overall mineralization level at a moderate level compared to the experimental groups.

[0133] Different composite culture media have a certain impact on the osteogenic differentiation ability of MSCs, and some culture media systems have shown good results in supporting the osteogenic differentiation of MSCs.

[0134] 3.7 BM-MSCs and AD-MSCs Proliferation Rate Following the same experimental procedure as in 3.5, fifth-generation BM-MSCs and AD-MSCs were seeded into 96-well plates at a cell density of 1 × 10⁶ cells per well. 4 Cells were cultured at 0.2 mL / well at 37°C and 5% CO2 for 7 days. CCK-8 reagent was added to each well at a ratio of 10% of the total culture medium volume and mixed thoroughly to prepare the CCK-8 detection working solution. Each day, the cell culture plate was removed, the original culture medium was discarded, and the cells were washed with PBS. CCK-8 detection solution was added to each well, and the plates were incubated under the same conditions for 2 hours. Subsequently, the optical density of each well was measured at 450 nm using a microplate reader to plot the cell growth curve.

[0135] like Figures 31-32 As shown, the in vitro proliferation capacity of AD-MSCs and BM-MSCs under different culture conditions was detected by the CCK-8 assay, and cell growth curves were plotted. The results showed that MSCs from both sources exhibited typical "S"-shaped growth curves under various culture conditions. In the early stage of culture (24-48 h), the cells were in the adaptation phase, with generally low OD values ​​and slow proliferation rates; from 48-96 h, the cells gradually entered the logarithmic growth phase, with significantly increased OD values ​​and a significantly accelerated cell proliferation rate; after 96 h, the cells gradually entered the plateau phase, and the rate of increase in OD values ​​gradually slowed down.

[0136] In the AD-MSCs culture system, the support for cell proliferation varied among different culture media. The OD value of the control group G (10% FBS medium) remained high throughout the culture period, indicating relatively stable cell proliferation. Some experimental groups, such as C, D, and H, showed significant increases in OD values ​​during the logarithmic growth phase, with cell proliferation trends similar to the control group. However, the OD values ​​of groups A, B, and E were relatively low, indicating slower cell proliferation. The growth curves of groups F, J, and I were at a moderate level, suggesting that different culture media had varying degrees of promoting effect on the in vitro expansion of AD-MSCs.

[0137] In the BM-MSCs culture system, cells in each group exhibited typical proliferation patterns. The control group (G) maintained a high OD value throughout the culture period, demonstrating good proliferation capacity. Some experimental groups, such as C, D, and H, showed significant OD value increases between 72 and 120 hours, with cell proliferation trends similar to the control group. Groups A, B, and E had relatively low OD values ​​and slower cell proliferation rates. Groups F, J, and I exhibited moderate proliferation capacity.

[0138] Comparing the growth curves of MSCs from the two sources reveals that both AD-MSCs and BM-MSCs can proliferate normally under the same culture medium conditions, but their proliferation rates and OD value trends differ. AD-MSCs show a larger increase in OD value during the logarithmic growth phase, indicating a faster proliferation trend, while the growth curve of BM-MSCs is relatively flat. The responses of MSCs from different sources to the culture medium components also differ.

[0139] 3.8 Monitoring of cloning capacity of BM-MSCs and AD-MSCs 9th generation BM-MSCs and AD-MSCs were seeded into 24-well plates at a cell density of 0.5-1 × 10⁶ cells per well. 3 Cells were cultured at a density of 10 cells / mL, with 0.2 mL of either the experimental group (AI) or the control group (G) culture medium added to each well. Cells were incubated continuously at 37℃ and 5% CO2 for 7–14 days. After incubation, cells were washed at least twice with PBS, and 250 μL of 4% paraformaldehyde was added to each well for fixation at room temperature for 15 min. The paraformaldehyde was discarded, and cells were washed twice with PBS. Then, 250 μL of 0.1% crystal violet was added to each well for staining at room temperature for 15 min. The crystal violet was discarded, and cells were rinsed thoroughly with running water and then air-dried upside down.

[0140] like Figure 33-36As shown, under 10 different culture medium conditions, MSCs from both sources formed distinct cell clones, which appeared as purple spots or clumps. Each culture system supported the adherent growth and clonal expansion of MSCs to some extent. Different numbers of clones were observed in each group of AD-MSCs. Groups D, E, and F formed relatively more cell clones, with denser distribution and some larger clones, demonstrating strong clone-forming ability. Groups A, B, and J had moderate clone numbers, while groups C, H, and I had relatively fewer clones with sparser distribution. The control group G (10% fetal bovine serum medium) also formed a certain number of cell clones, with an overall clone density at a moderate level compared to the experimental groups. The clone-forming results of BM-MSCs were similar to those of AD-MSCs, but the degree to which different culture media promoted clone-forming ability varied. Some experimental groups also showed high colony formation capacity. Among them, groups B, C, and E formed a large number of colonies with relatively uniform distribution. These culture conditions provided good support for the proliferation potential of BM-MSCs. Groups A and D had the next highest number of colonies, while groups F and H had relatively fewer colonies. The control group G formed stable colonies, but the overall density was slightly lower than that of some of the superior experimental groups.

[0141] Comparing the clonal formation of MSCs from two sources, different composite culture media all had a certain impact on the cell clonal formation ability. Among them, the culture systems of groups A, E and F showed better effects in promoting the clonal formation of AD-MSCs and BM-MSCs. These culture conditions have certain advantages in maintaining the proliferation potential of MSCs.

[0142] 3.9 Monitoring of cellular senescence in BM-MSCs and AD-MSCs BM-MSCs and AD-MSCs were cultured to the ninth generation using AI medium (experimental group) and G medium (control group), respectively. The cells were then passaged into 24-well plates and cultured for 24 h. After washing with PBS, 250 μL of β-galactosidase staining solution was added to fix the cells at room temperature for 15 min. After removing the cell fixative, the cells were washed with PBS at least three times for 3 min each time. 250 μL of β-galactosidase staining solution was added to each well. The plates were sealed with film to prevent CO2 from entering and incubated overnight at 37°C.

[0143] like Figures 37-38As shown, microscopic observation revealed varying degrees of senescent positive cells in both AD-MSCs and BM-MSCs cultured under 10 different culture medium conditions. However, most cells maintained typical spindle-shaped or fibroblast-like adherent growth morphology, with relatively uniform cell arrangement. Each culture system effectively maintained the in vitro cell proliferation state to some extent. In AD-MSCs, the degree of senescence varied among the experimental groups. Groups B, E, and F had relatively fewer blue-stained positive cells, with more uniform cell morphology and higher density. Groups C, D, and G showed a certain proportion of senescent positive cells, indicating a moderate level of senescence. Groups A, H, and I had a relatively larger number of blue-stained cells, with some cells increasing in size and exhibiting a flattened morphology, suggesting a relatively higher degree of cell senescence. The control group G (10% FBS medium) also showed a certain number of senescent positive cells, with an overall senescence level similar to some experimental groups. In BM-MSCs, cell senescence under different culture medium conditions showed a trend similar to, but slightly different from, that of AD-MSCs. Cells in groups B, E, and F showed relatively uniform morphology and a relatively low proportion of senescent positive cells. A certain number of blue-stained positive cells were observed in groups C, D, and J. Groups A, H, and I showed a relatively high proportion of senescent cells, with some cells exhibiting increased size and flattened morphology. The control group G also showed a certain degree of senescence-positive signal.

[0144] AD-MSCs and BM-MSCs showed similar senescence trends under different culture conditions. The senescence of cells in the B, E and F groups was relatively low, while the senescence of cells in the A, H and I groups was more obvious.

[0145] This embodiment established a bPL-based MSCs system and optimized the corresponding culture conditions to provide a stable foundation for supporting the in vitro culture and expansion of MSCs. Using BM-MSCs and AD-MSCs as research subjects, systematic cell morphology observation, CCK-8 proliferation detection, colony formation assay, SA-β-gal senescence detection, and adipogenic and osteogenic induction differentiation experiments were conducted. A comprehensive evaluation system for the proliferation capacity, senescence level, and multi-lineage differentiation potential of mesenchymal stem cells was established. Through comparative analysis and comprehensive evaluation of different culture systems, the results showed that the bPL-based culture system could stably support the in vitro proliferation of BM-MSCs and AD-MSCs and maintain their multi-lineage differentiation capacity. Groups E and F (5% sonication bPL, 10% sonication, and 10% Ga²⁺ activation method) showed similar or even better culture effects to the FBS culture system in terms of cell proliferation capacity and senescence level, providing experimental evidence for the application of bPL in the in vitro culture and serum replacement studies of MSCs.

[0146] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A bovine platelet lysis buffer, characterized in that, The bovine platelet lysate is produced by repeatedly freezing and thawing bovine platelets, ultrasonically disrupting them, or using Ga... 2+ It is prepared by pyrolysis through activation.

2. A method for preparing bovine platelet lysis buffer, characterized in that, The method involves repeatedly freezing and thawing bovine platelets, ultrasonically disrupting them, or subjecting them to Ga... 2+ The bovine platelet lysate was obtained by activation treatment.

3. The method according to claim 2, characterized in that, The repeated freeze-thaw cycles consisted of three freeze-thaw cycles at -80℃ and 37℃, followed by centrifugation at 4000 rpm for 30 min, and filtration of the supernatant through a 0.22 μm filter. The ultrasonic disruption consisted of ten cycles at 285 W, with each cycle lasting 5 seconds followed by a 5-second pause; centrifugation at 4000 rpm for 30 min followed by filtration of the supernatant through a 0.22 μm filter. Ga 2+ Activation is: adding Ga to platelets. 2+ The calcium chloride solution with a final concentration of 4.17 mmol / L was mixed thoroughly, allowed to stand in a water bath for 5 min, centrifuged at 4000 rpm for 30 min, and the supernatant was collected and filtered through a 0.22 μm filter.

4. The method according to claim 2, characterized in that, The bovine platelet lysate is stored at a temperature below -20°C.

5. A compound formulation culture medium, characterized in that, The compound culture medium contains bovine platelet lysate prepared by the method of claim 2.

6. The compound culture medium according to claim 5, characterized in that, The compound culture medium has undergone fibrinogen depletion treatment.

7. The compound formulation culture medium according to claim 5, characterized in that, The compound culture medium, by weight, contains 2% ultrasonically disrupted bovine platelet lysate + 8% adult bovine serum, 4% repeatedly freeze-thawed bovine platelet lysate + 6% adult bovine serum, or 4% ultrasonically disrupted bovine platelet lysate + 6% adult bovine serum.

8. The application of the compound culture medium according to any one of claims 5-7 in cell proliferation and cell cloning.

9. A mesenchymal stem cell culture medium, characterized in that, The mesenchymal stem cell culture medium comprises bovine platelet lysate prepared by the method of claim 2 and DMEM.

10. The use of the culture medium according to claim 9 in adipogenic differentiation, osteogenic differentiation, supporting cell proliferation in vitro, or maintaining multi-lineage differentiation capacity.

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